CN110144399A - Detect primer sets, kit and the application method of lung cancer related gene mutation in mankind's Circulating tumor DNA - Google Patents
Detect primer sets, kit and the application method of lung cancer related gene mutation in mankind's Circulating tumor DNA Download PDFInfo
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Abstract
The invention discloses primer sets, kit and the application methods of lung cancer related gene mutation in a kind of detection mankind's Circulating tumor DNA, 49 for providing 5 genes suffer from the specificity of lung-cancer-risk and the genetic mutation site of sensibility for assessing the mankind, carry out gene analysis test to these sites using MassARRAY nucleic acid mass-spectrometric technique.The ddNTPs for being used for elongation mutagenesis type gene is only added when this kit Single base extension, and ddNTPs has biotin labeling, extension products are captured using the magnetic bead with marked by streptavidin, remove the signal interference of miscellaneous peak, the signal value for greatly increasing mutated genes in extension products improves the sensitivity of kit.Circulating tumor DNA sample is detected using this method, its sensitivity of 20ng applied sample amount can be down to 0.1%.The method compares qPCR and two generation microarray datasets, has the advantages such as flexible design, accuracy be high, at low cost, easy to operate.
Description
Technical field
The invention belongs to genetic test fields, more particularly, to lung cancer dependency basis in a kind of detection mankind's Circulating tumor DNA
Because of the primer sets of mutation, kit and application method, using multiple PCR technique, Single base extension technology and nucleic acid mass-spectrometric technique,
5 genes relevant to lung cancer, 49 sites carry out genetic test.
Background technique
Tumour is that polygenes experience multi-step variation leads to cell cycling disorder, and uncontrolled cellular is made to grow and be formed new
Growth-gen.Lung cancer is to seriously threaten one of malignant tumour of human health, wherein non-small cell lung cancer (non-small cell
Lung cancer, NSCLC) account in 85% of lung cancer or so, NSCLC it is again most common with gland cancer.It is more in oncology, science of heredity etc.
It is found in disciplinary study, there are EGF-R ELISA (Epidermal Growth Factor by most of NSCLC patients
Receptor, EGFR) etc. proto-oncogenes and tumor suppressor gene mutation, they tumour formation and its mediate oncocyte biology
It plays an important role in behavior, therefore these genes become one of the important target spot of antineoplastic target therapeutic agent.
The effective percentage of cancer clinical treatment is still relatively low at present, this is poor with the genesis mechanism of cancer complexity and individual
It is different closely related.Deepening continuously and develop with human activities environment, pharmacogenomics and Cancer Molecular biological study,
Research shows that in human body the targeting of certain genes and cancer or the curative effect of chemotherapeutics or toxicity be it is closely related, to these spy
Determine gene to be detected, be briefly exactly the histocyte, blood or pleural effusion for taking detected person, it is extracted and purify its
After gene information, the specific gene in detected person's cell is detected by particular device, the state for analyzing gene is cancer
Patient's offer therapeutic scheme reference is more scientific, helps to improve quality of life and the life cycle of patient, and save patient's
Time and money.
Some researches show that be can detecte in the body fluid such as blood, pleural effusions and ascites, cerebrospinal fluid of tumor patient is free
DNA content is much larger than normal person, and dissociative DNA shows biological characteristics identical with tumor tissues.With tumor cells
The progress of biological study, the detection of dissociative DNA and its research of biological indicator have been that clinical tumor early diagnoses, prognosis is sentenced
Disconnected and tracking follow-up etc. provides a series of convenient, fast, specially, noninvasive or minimally invasive molecular Biological Detection means.
Nucleic acid mass spectrum detection combines round pcr, Single base extension technology, passes through PCR amplification amplification detection sample
This template quantity；Extend a base using the Single base extension technology specificity of the title with " micro sequence ", accuracy is high；Especially
It is the molecular weight that Mass Spectrometer Method target is substance, is marked without fluorescence probe, resolution ratio when identifying the difference between single base
Up to 9Da, so that the technology has very vigorous vitality in genetic test field.The technology won Nobel from 2002
Since prize, by all circles' extensive concern, have numerous applications in various fields such as medicine, agriculturals.
Summary of the invention
The present invention provides the primer sets that lung cancer related gene in one group of detection mankind's Circulating tumor DNA makes a variation.
It is a further object to provide lung cancer related gene variation reagents in a kind of detection mankind's Circulating tumor DNA
Box, the kit can detect multiple mutational sites simultaneously, and flux is high.Detection time is shorter, and the expense of single locus is relatively
It is low.
Third object of the present invention is to provide a kind of examinations of lung cancer related gene variation in detection mankind's Circulating tumor DNA
The application method of agent box.Sensitivity for analysis and analysis specificity are good, easy to operate.
Unlike common nucleic acid Mass Spectrometer Method product, only it is added when this kit Single base extension and is used for elongation mutagenesis
The ddNTPs of type gene, and ddNTPs has biotin labeling, is produced using the magnetic bead with marked by streptavidin to extension
Object is captured, and the signal interference of miscellaneous peak is removed, to considerably increase the signal value of mutated genes in extension products, is improved
The sensitivity of kit.Circulating tumor DNA sample is detected using this method, its sensitivity of 20ng applied sample amount can be down to 0.1%.
The method compares qPCR and two generation microarray datasets, has the advantages such as flexible design, accuracy be high, at low cost, easy to operate.
In order to solve the above-mentioned technical problem, the technical solution adopted by the present invention is that: in a kind of detection mankind's Circulating tumor DNA
The primer sets of lung cancer related gene mutation, primer sets include PCR amplification primer, Single base extension primer and quality control of procedure primer,
PCR amplification primer sequence is SEQ ID No.1-22, Single base extension primer sequence are as follows: SEQ ID No.23-62, quality control of procedure
Primer sequence are as follows: SEQ ID No.63-65.
The Single base extension primer is divided into 8 groups:
A kind of kit detecting Human Lung Cancer associated gene mutation, comprising:
1) reaction reagent of PCR, including PCR amplification primer described in SEQ ID No.1-22, SEQ ID No.63-64 are used for
The quality control of procedure primer, archaeal dna polymerase resistant to high temperature, dNTPs, MgCl2, PCR reaction buffer；
2) reagent for PCR product purifying, including shrimp alkaline phosphotase, shrimp alkaline phosphotase buffer；
3) reagent of Single base extension, the Single base extension primer including SEQ ID No.23-62, SEQ ID are used for
The quality control of procedure primer of No.65, single base extension enzyme resistant to high temperature, biotin labeling ddNTPs, extension buffer solution.
4) reagent for extension products capture, the magnetic bead including marked by streptavidin, in conjunction with/washing buffer.
Kit further include: negative quality-control product, positive quality control product capture quality-control product, purifying resin, point sample and mass spectrum inspection
The target plate of survey.
The specificity of lung-cancer-risk and the gene mutation site of sensibility are suffered from for assessing the mankind are as follows: BRAF_
p.D594G、BRAF_p.G469A、BRAF_p.G469V、BRAF_p.V600E、EGFR_p.C797S、EGFR_p.D770_
N771insSVD、EGFR_p.D770_N771insG、EGFR_p.E709A、EGFR_p.E709G、EGFR_p.E709K、EGFR_
P.E746_A750del, EGFR_p.E746_E749del, EGFR_p.E746_S752 > D, EGFR_p.E746_S752 > V,
EGFR_p.E746_T751del、EGFR_p.G719A、EGFR_p.G719C、EGFR_p.G719S、EGFR_p.H773_
V774insNPH, EGFR_p.H773_V774insH, EGFR_p.L747_A750 > P, EGFR_p.L747_E749del, EGFR_
P.L747_P753 > S, EGFR_p.L858R, EGFR_p.L861Q, EGFR_p.L861R, EGFR_p.S768I, EGFR_
P.T790M, EGFR_p.V769_D770insASV, ERBB2_p.A775_G776insYVMA, ERBB2_p.G776 > VC,
KRAS_p.G12A、KRAS_p.G12V、KRAS_p.G12D、KRAS_p.G12R、KRAS_p.G12C、KRAS_p.G12S、KRAS_
p.G13C、KRAS_p.G13D、KRAS_p.Q61H、KRAS_p.Q61K、KRAS_p.Q61E、KRAS_p.Q61P、KRAS_
p.Q61L、KRAS_p.Q61R、PIK3CA_p.E542K、PIK3CA_p.E545K、PIK3CA_p.H1047L、PIK3CA_
p.H1047R。
The application method of the kit of above-mentioned detection Human Lung Cancer associated gene mutation, includes the following steps:
1) multi-PRC reaction: in a reaction system, using sample to be tested DNA as template, using specificity for packet
PCR amplification primer containing detected gene mutation site expands the sequence containing different gene locis to be checked by multiplex PCR simultaneously
Column obtain the PCR product of the target area containing amplification；
2) shrimp alkaline phosphatase enzymatic digestion stage 1 is utilized) remaining dNTPs in reaction system；
3) single base extension is carried out using the postdigestive product of Single base extension primer pair step 2), obtains and extends production
Object；
4) extension products of saltant type in the magnetic capture single base extension product of marked by streptavidin are utilized；
5) purifying resin, chip point sample, Mass Spectrometer Method are carried out using MassARRAY platform, it is as a result soft using TYPER4.0
Part is analyzed and is exported as a result, by the way that whether appearance judges mutation type at variation base.
The PCR reaction condition of the step 1) are as follows: 95 DEG C, 2min；95 DEG C, 30s, 56 DEG C, 30s, 72 DEG C, 1min, 45
Circulation；72℃,5min；4 DEG C of holdings.
The digestion condition of the step 2) are as follows: 37 DEG C, 40min；85℃,5min；4 DEG C of holdings.
The extension condition of the step 3) are as follows: 95 DEG C, 30s；95 DEG C, 5s, (52 DEG C, 5s, 80 DEG C, 5min) 5 follow
Ring, 40 circulations；72℃,3min；4 DEG C of holdings.
The beneficial effects of the present invention are:
1) high sensitivity.The present invention combines multiple PCR technique, Single base extension technology, mass-spectrometric technique and is integrated, and can lead to
Multiple PCR technique amplification template is crossed, and trace sample can be detected by mass-spectrometric technique, improves the sensitivity of detection.It compares general
For logical nucleic acid mass-spectrometric technique, the ddNTPs for being used for elongation mutagenesis type gene is only added in when this kit Single base extension, and
DdNTPs has biotin labeling, is captured using the magnetic bead with marked by streptavidin to extension products, removes miscellaneous peak
Signal interference improve the sensitivity of kit to considerably increase the signal value of mutated genes in extension products.Needle
To plasma DNA, 20ng applied sample amount can detect the mutation down to 0.1%.
2) specificity is good.Single base extension only extends a base in 3 ' end of primer using specific extension primer, compared with
For sequencing technologies extend hundreds of bases, error probability is lower, and accuracy is high, and specificity is good.
3) present invention can simultaneously detect 5 genes, 49 sites of multiple samples, for comparing qPCR technology
Flux is higher.
4) quick, easy.The present invention can complete detection, easy to operate, high degree of automation in one day, and be not necessarily to complexity
Bioinformatic analysis.
5) relative to other detection methods, cost is relatively low for single locus.Alleviate the medical treatment burden of patient.
6) sample input amount is less.Sample is relatively saved.
7) peripheral blood sample is used, the great pain that patient is subject to by tissue penetration is avoided.
8) kit of the invention can instruct the targeting medication of patient, help to improve the quality of life and existence of patient
Phase clinically has great importance.
Specific embodiment
Invention is further described in detail combined with specific embodiments below.
The primer sets of lung cancer related gene mutation in detection mankind's Circulating tumor DNA of the invention, primer sets include that PCR expands
Increase primer, Single base extension primer and quality control of procedure primer, PCR amplification primer sequence is SEQ ID No.1-22, and single base is prolonged
Stretch primer sequence are as follows: SEQ ID No.23-62, quality control of procedure primer sequence are as follows: SEQ ID No.63-65.
It is preferred that the Single base extension primer is divided into 8 groups:
A kind of kit detecting Human Lung Cancer associated gene mutation, comprising:
1) reaction reagent of PCR, including PCR amplification primer described in SEQ ID No.1-22, SEQ ID No.63-64 are used for
The quality control of procedure primer, archaeal dna polymerase resistant to high temperature, dNTPs, MgCl2, PCR reaction buffer；
2) reagent for PCR product purifying, including shrimp alkaline phosphotase, shrimp alkaline phosphotase buffer；
3) reagent of Single base extension, the Single base extension primer including SEQ ID No.23-62, SEQ ID are used for
The quality control of procedure primer of No.65, single base extension enzyme resistant to high temperature, biotin labeling ddNTPs, extension buffer solution.
4) reagent for extension products capture, the magnetic bead including marked by streptavidin, in conjunction with/washing buffer.
Kit further include: negative quality-control product, positive quality control product capture quality-control product, purifying resin, point sample and mass spectrum inspection
The target plate of survey.
The specificity of lung-cancer-risk and the gene mutation site of sensibility are suffered from for assessing the mankind are as follows: BRAF_
p.D594G、BRAF_p.G469A、BRAF_p.G469V、BRAF_p.V600E、EGFR_p.C797S、EGFR_p.D770_
N771insSVD、EGFR_p.D770_N771insG、EGFR_p.E709A、EGFR_p.E709G、EGFR_p.E709K、EGFR_
P.E746_A750del, EGFR_p.E746_E749del, EGFR_p.E746_S752 > D, EGFR_p.E746_S752 > V,
EGFR_p.E746_T751del、EGFR_p.G719A、EGFR_p.G719C、EGFR_p.G719S、EGFR_p.H773_
V774insNPH, EGFR_p.H773_V774insH, EGFR_p.L747_A750 > P, EGFR_p.L747_E749del, EGFR_
P.L747_P753 > S, EGFR_p.L858R, EGFR_p.L861Q, EGFR_p.L861R, EGFR_p.S768I, EGFR_
P.T790M, EGFR_p.V769_D770insASV, ERBB2_p.A775_G776insYVMA, ERBB2_p.G776 > VC,
KRAS_p.G12A、KRAS_p.G12V、KRAS_p.G12D、KRAS_p.G12R、KRAS_p.G12C、KRAS_p.G12S、KRAS_
p.G13C、KRAS_p.G13D、KRAS_p.Q61H、KRAS_p.Q61K、KRAS_p.Q61E、KRAS_p.Q61P、KRAS_
p.Q61L、KRAS_p.Q61R、PIK3CA_p.E542K、PIK3CA_p.E545K、PIK3CA_p.H1047L、PIK3CA_
p.H1047R。
The application method of the kit of above-mentioned detection Human Lung Cancer associated gene mutation, includes the following steps:
1) multi-PRC reaction: in a reaction system, using sample to be tested DNA as template, using specificity for packet
PCR amplification primer containing detected gene mutation site expands the sequence containing different gene locis to be checked by multiplex PCR simultaneously
Column obtain the PCR product of the target area containing amplification；
2) shrimp alkaline phosphatase enzymatic digestion stage 1 is utilized) remaining dNTPs in reaction system；
3) single base extension is carried out using the postdigestive product of Single base extension primer pair step 2), obtains and extends production
Object；
4) extension products of saltant type in the magnetic capture single base extension product of marked by streptavidin are utilized；
5) purifying resin, chip point sample, Mass Spectrometer Method are carried out using MassARRAY platform, it is as a result soft using TYPER4.0
Part is analyzed and is exported as a result, by the way that whether appearance judges mutation type at variation base.
The PCR reaction condition of the step 1) are as follows: 95 DEG C, 2min；95 DEG C, 30s, 56 DEG C, 30s, 72 DEG C, 1min, 45
Circulation；72℃,5min；4 DEG C of holdings.
The digestion condition of the step 2) are as follows: 37 DEG C, 40min；85℃,5min；4 DEG C of holdings.
The extension condition of the step 3) are as follows: 95 DEG C, 30s；95 DEG C, 5s, (52 DEG C, 5s, 80 DEG C, 5min) 5 follow
Ring, 40 circulations；72℃,3min；4 DEG C of holdings.
The principle of the invention lies in: provide a kind of joint multiple PCR technique, Single base extension technology and Mass Spectrometer Method skill
Art, the method for detecting Human Lung Cancer associated gene mutation.Wherein, it is expanded in multiplex PCR up to containing 49 lung cancer related genes
The DNA fragmentation in mutational site；During Single base extension, the ddNTPs for being used for elongation mutagenesis type gene is only added, and
DdNTPs has biotin labeling, is captured using the magnetic bead with marked by streptavidin to extension products, removes miscellaneous peak
Signal interference improve the sensitivity of kit to considerably increase the signal value of mutated genes in extension products；It is single
When base extends, multiple Single base extension is carried out to the purified product of multiplex PCR, single base extension product is punished in 49 sites
Not Yan Shen a base so that the base type extended is related to the genotype in the site respectively；Single base extension generate by
The mixture to be checked of extension primer and extension products composition, detects mixture to be checked with mass spectrographic method, with molecular weight
To distinguish every kind of molecule in product to be checked, and carried out with the theoretical molecular weight of each extension primer and extension products that precalculate
It compares, so that it is determined that the genotype in each site.
Specifically the present invention includes PCR amplification for detecting the primer sets in 5 mankind's lung cancer related genes, 49 sites
Primer, Single base extension primer and quality control of procedure primer, sequence are as shown in table 1.
Table 1
Wherein, 49 gene mutation sites for being used to assess specificity and sensibility that the mankind suffer from lung-cancer-risk are
BRAF_p.D594G、BRAF_p.G469A、BRAF_p.G469V、BRAF_p.V600E、EGFR_p.C797S、EGFR_p.D770_
N771insSVD、EGFR_p.D770_N771insG、EGFR_p.E709A、EGFR_p.E709G、EGFR_p.E709K、EGFR_
P.E746_A750del, EGFR_p.E746_E749del, EGFR_p.E746_S752 > D, EGFR_p.E746_S752 > V,
EGFR_p.E746_T751del、EGFR_p.G719A、EGFR_p.G719C、EGFR_p.G719S、EGFR_p.H773_
V774insNPH, EGFR_p.H773_V774insH, EGFR_p.L747_A750 > P, EGFR_p.L747_E749del, EGFR_
P.L747_P753 > S, EGFR_p.L858R, EGFR_p.L861Q, EGFR_p.L861R, EGFR_p.S768I, EGFR_
P.T790M, EGFR_p.V769_D770insASV, ERBB2_p.A775_G776insYVMA, ERBB2_p.G776 > VC,
KRAS_p.G12A、KRAS_p.G12V、KRAS_p.G12D、KRAS_p.G12R、KRAS_p.G12C、KRAS_p.G12S、KRAS_
p.G13C、KRAS_p.G13D、KRAS_p.Q61H、KRAS_p.Q61K、KRAS_p.Q61E、KRAS_p.Q61P、KRAS_
p.Q61L、KRAS_p.Q61R、PIK3CA_p.E542K、PIK3CA_p.E545K、PIK3CA_p.H1047L、PIK3CA_
p.H1047R。
Wherein, extension primer is divided into 8 groups according to each site different molecular weight and genotype, specific to be grouped situation such as 2 institute of table
Show.
Table 2
In one embodiment, tag (the 5 '-ACGTTGGATG- of 10bp are added at 5 ' ends in above-mentioned PCR primer sequence
3').For example, PCR primer SEQ ID No.1 sequence is 5 '-ACGTTGGATGTTACCTTATACACCGTGCCG-3 '.
Technology detects lung cancer related gene mutational site to embodiment one through the invention
One, design of primers and synthesis
For BRAF_p.D594G, BRAF_p.G469A, BRAF_p.G469V, BRAF_p.V600E, EGFR_p.C797S,
EGFR_p.D770_N771insSVD、EGFR_p.D770_N771insG、EGFR_p.E709A、EGFR_p.E709G、EGFR_
P.E709K, EGFR_p.E746_A750del, EGFR_p.E746_E749del, EGFR_p.E746_S752 > D, EGFR_
P.E746_S752 > V, EGFR_p.E746_T751del, EGFR_p.G719A, EGFR_p.G719C, EGFR_p.G719S,
EGFR_p.H773_V774insNPH, EGFR_p.H773_V774insH, EGFR_p.L747_A750 > P, EGFR_p.L747_
E749del, EGFR_p.L747_P753 > S, EGFR_p.L858R, EGFR_p.L861Q, EGFR_p.L861R, EGFR_
p.S768I、EGFR_p.T790M、EGFR_p.V769_D770insASV、ERBB2_p.A775_G776insYVMA、ERBB2_
P.G776 > VC, KRAS_p.G12A, KRAS_p.G12V, KRAS_p.G12D, KRAS_p.G12R, KRAS_p.G12C, KRAS_
p.G12S、KRAS_p.G13C、KRAS_p.G13D、KRAS_p.Q61H、KRAS_p.Q61K、KRAS_p.Q61E、KRAS_
p.Q61P、KRAS_p.Q61L、KRAS_p.Q61R、PIK3CA_p.E542K、PIK3CA_p.E545K、PIK3CA_p.H1047L、
PIK3CA_p.H1047R.It is used to assess the mankind Deng 49 and suffers from the specificity of lung-cancer-risk and the gene mutation site of sensibility,
Design corresponding Specific PCR primers core sequence (SEQ ID No.1 to SEQ ID No.22) and specific extension primer
(SEQ ID No.23 to SEQ ID No.62).
Wherein, in order to avoid PCR primer enter mass spectrograph detection window and interference detection results, in every PCR primer
5 ' ends introduce the base of certain amounts, such as the tag (ACGTTGGATG) of 10bp, to increase the molecular weight of every PCR primer, thus
More than the window of mass spectrograph detection, avoid influencing result interpretation.
Two, sample DNA extracts
DNA extraction is carried out using commercialized nucleic acid extraction kit (Ai De biotinylated nucleic acid extracts reagent (Circulating DNA)).
The DNA of extraction is quantified with Qubit (Thermo company).DNA after extraction as required, is saved small no more than 24 at 2~8 DEG C
When, -15~-25 DEG C save be no more than 2 years, -75~-85 DEG C can long-term preservation, multigelation should be avoided, if you need to be transported,
It please use ice chest.
1, sample is plasma sample, requires to specifications, should ensure that haemolysis does not occur for sample.Extraction step by specification
It is operated.
2, sample is DNA sample, and after quality inspection is qualified, -15~-25 DEG C are saved backup.
Three, Mass Spectrometer Method
(1) pcr amplification reaction
1 prepares a 1.5ml centrifuge tube, and according to sample size, according to the form below prepares PCR reaction solution.PCR reaction solution each component
It is shown in Table 4:
Table 4
| Ingredient names | Single reaction volume (μ L) |
| Hplc grade water | 8.05 |
| PCR reaction buffer | 7.00 |
| MgCl2 | 2.80 |
| dUTP/dNTP Mix | 0.35 |
| PCR amplification primer | 14.00 |
| High temperature-resisting DNA polymerase | 2.80 |
| PCR reaction solution total volume | 35.00 |
2 prepare 96 orifice plates, are dispensed above-mentioned PCR reaction solution into reacting hole according to sample size, every 35 μ L of hole.
3 the DNA sample diluted is added into the reacting hole for dispensed PCR reaction solution, makes the final usage amount of sample
20ng, PCR react 70 μ L of total volume, insufficient plus water polishing.
4 will add excellent 96 orifice plate to be placed in PCR amplification instrument, carry out pcr amplification reaction by the program of table 5.
Table 5
(2) shrimp alkaline phosphotase digests
1 digests the product of PCR amplification using shrimp alkaline phosphotase, prepares digestion reaction liquid according to sample number, disappears
Change reaction solution each component and is shown in Table 6
Table 6
| Ingredient names | Single reaction volume (μ L) |
| Hplc grade water | 21.42 |
| Shrimp alkaline phosphotase buffer | 2.38 |
| Shrimp alkaline phosphotase | 4.20 |
| Digestion reaction liquid total volume | 28.00 |
2 as being added digestion reaction liquid, every 28 μ L of hole in 96 orifice plates.
3 are placed in 96 orifice plates in PCR instrument, carry out digestion process to PCR product by the program of table 7.
Table 7
(3) Single base extension
Digestion product in 1 pair of step 2 carries out Single base extension, corresponding 8 extensions of a digestion product of each sample
Reaction.1 1.5mL centrifuge tube is taken, single base extension liquid 1 is prepared according to sample number, reaction solution each component is shown in Table 8.
Table 8
| Ingredient names | Single reaction volume (μ L) |
| Hplc grade water | 0.519 |
| Extension buffer solution | 0.200 |
| Single base extension enzyme | 0.041 |
| Capture quality-control product | 0.100 |
| 1 total volume of extension liquid | 0.860 |
2 are sub-packed in configured extension liquid 1 in 8 PCR pipes according to sample number, number 1-8.
3 separately take five new 1.5mL centrifuge tubes, prepare Termination mix, this Termination Mix according to table 9
For the use of entire Panel kit, 2~8 DEG C of storages are placed when not used.
Table 9
4 prepare extension liquid E1-E8 according to sample size, by table 10.Wherein Termination Mix and extension primer
It is one-to-one relationship, corresponding relationship is shown in Table 11.
Table 10
| Ingredient names | Single reaction volume (μ L) |
| Extension liquid 1 | 0.86 |
| Extension primer (E1-E8) | 0.94 |
| Termination Mix | 0.20 |
| Extension liquid E1-E8 | 2.00 |
Table 11
5 separately take a 96 new orifice plates, sample each in the digestion product of step 2 are assigned in 8 reacting holes, every hole 7
μL。
6 are added to extension liquid E1-E8 in the 1-8 reacting hole of each sample correspondingly, every 2 μ L of hole.
7 are placed in 96 orifice plates in PCR instrument, carry out single base extension by the program of table 12.
Table 12
(4) Streptavidin MagneSphere captures
1 gets out 1.5mL centrifuge tube, calculates by the magnetic bead of each 4.25 μ L of reacting hole.
2 notes: Streptavidin MagneSphere must be saved at 4 DEG C, be mixed using preceding with pipettor pressure-vaccum.
3 place centrifuge tube on magnetic frame, siphon away storing liquid (not destroy magnetic bead) using 1mL pipette tips after 1 minute.
4 are added combination/cleaning buffer solution of same volume, simultaneously brief centrifugation (guaranteeing no liquid on lid) are resuspended, then will
Centrifuge tube is placed on magnetic frame, and combination/cleaning buffer solution is siphoned away after 1 minute.
5 repeat step 4 twice.
6 take down centrifuge tube from magnetic frame.Using the centrifuge tube of 5mL, by each reaction (stoichiometric number is identical as step 1) 25
Combination/cleaning buffer solution of μ L addition respective volume.
7 are added hplc grade water by each reaction (stoichiometric number is identical as step 1) 16 μ L and are resuspended.
8 are transferred to the magnetic bead after resuspension in V-shaped groove from 5mL centrifuge tube.
9 magnetic bead, each 41 μ L of hole (each 50 μ L of reaction total volume) are added into 96 orifice plates with the volley of rifle fire.
10 96 orifice plates of sealing, are placed on board-like rotator, are incubated at room temperature at least 30min.
11 have been incubated for centrifugation, and 96 orifice plates are placed on board-like magnetic frame, supernatant is removed after 1min.
12 take down reaction plate from magnetic frame, and 100 μ L hplc grade waters, pressure-vaccum is added into each reacting hole with the volley of rifle fire
It mixes at least 8 times, throws away pipette tips.Guarantee that magnetic bead is resuspended completely.
96 orifice plates are placed on board-like magnetic frame, supernatant are removed after 1min by 13 brief centrifugations.
14 repetition step 12-13 are primary.
15 take down reaction plate from magnetic frame.Biotin solution is poured into V-shaped groove, with the volley of rifle fire to each hole of 96 orifice plates
13 μ L biotin solutions of middle addition.
16 sealing reaction plates, be vortexed 5 resuspension magnetic beads of concussion, and rear of short duration centrifugation, which is placed in PCR instrument, carries out following thermal cycle:
Table 13
| Temperature | Time | Recurring number |
| 90℃ | 5minutes | 1 |
| 10℃ | Hold |
After 17 have reacted, it is centrifuged 1000 × g of reaction plate 1min.Solution in transfer reaction plate is into 96 new orifice plates.
(5) machine testing and result interpretation on
1 is placed in 96 orifice plates in mass spectrograph, carries out purifying resin using MassARRAY Chip prep module system
And chip point sample.
Mass Spectrometer Method is carried out using MassARRAY Analyzer after 2 point samples.
3 as shown in aforementioned table 3, and every extension primer and the extension products that they generate on different genes site all have
Different molecular weight, these molecular weight correspond to different mass spectra peaks, if there is mass spectra peak in certain molecular weight, be judged as in the presence of with
The corresponding extension primer of the molecular weight or product.
Judgment criteria:
Capture quality-control product five different peaks of molecular weight should occur, be respectively as follows: 4694.2Da, 4998.4Da, 9316.2Da,
9620.4Da,7134.8Da.A peak should occur in quality control of procedure product (quality control of procedure product are without peak in NTC).
1) mass spectra peak for the mass spectra peak and quality control of procedure product for capturing quality-control product has one kind not occur, and no matter extension products peak goes out
Whether now, it is judged as the failure of an experiment.
2) if the corresponding mass spectra peak of saltant type occurs, it is judged as the corresponding saltant type in the peak.
3) if the corresponding mass spectra peak of saltant type does not occur, it is judged as wild type.
Embodiment two: standard items detection
9 standard items DNA samples (being purchased from Horizon company) of known mutations are selected to be detected, number A1-A9.Its
In, it is wild type that the corresponding every kind of mutational site frequency of mutation of each sample of A1-A8, which is 0.1%, A9,.
According to the method for the present invention, according to step described in embodiment one, mass spectrum is carried out to 9 standard items DNA samples
Detection.Interpretation of result is carried out to 9 standard items samples according to a kind of criterion of description of embodiment, obtains table 14.
Table 14
Testing result, mutation corresponding to 8 saltant type standard items samples can be detected normally, illustrate the method for the present invention energy
20ng applied sample amount is detected down to the site of 0.1% frequency of mutation, there is forward position advantage.
Embodiment three: dissociative DNA pattern detection
According to the method for the present invention, according to step described in embodiment one, to the dissociative DNA sample of 30 patients with lung cancer
Carry out Mass Spectrometer Method, sample number B1-B30.30 samples are tied according to a kind of criterion of description of embodiment
Fruit analysis, obtains table 15.
Table 15
| Sample | The mutation of detection | Sample | The mutation of detection |
| B1 | KRAS_p.G12D | B16 | EGFR_p.S768I |
| B2 | KRAS_p.G12C | B17 | EGFR_p.L858R |
| B3 | EGFR_p.G719S、EGFR_p.L861Q | B18 | ERBB2_p.A775_G776insYVMA |
| B4 | EGFR_p.G719S、EGFR_p.L861Q | B19 | KRAS_p.G12S |
| B5 | EGFR_p.T790M | B20 | EGFR_p.E746_A750del |
| B6 | EGFR_p.E746_A750del | B21 | KRAS_p.G12D |
| B7 | BRAF_p.V600E | B22 | EGFR_p.V769_D770insASV |
| B8 | EGFR_p.S768I、EGFR_p.L858R | B23 | EGFR_p.E746_A750del |
| B9 | EGFR_p.L861Q | B24 | EGFR_p.S768I、EGFR_p.L858R |
| B10 | EGFR_p.L858R | B25 | KRAS_p.G12V |
| B11 | EGFR_p.L858R | B26 | EGFR_p.E746_A750del |
| B12 | KRAS_p.G12S | B27 | EGFR_p.L858R |
| B13 | BRAF_p.V600E | B28 | EGFR_p.L858R |
| B14 | PIK3CA_p.H1047R | B29 | BRAF_p.V600E |
| B15 | EGFR_p.L858R | B30 | KRAS_p.G12S、EGFR_p.L858R |
In 30 patients, altogether detect BRAF_p.V600E be mutated 3, EGFR_p.E746_A750del be mutated 4,
EGFR_p.G719S is mutated 2, EGFR_p.L861Q mutation 3, EGFR_p.L858R mutation 9, EGFR_p.S768I mutation 3
Example, EGFR_p.T790M are mutated 1, EGFR_p.V769_D770insASV mutation 1, ERBB2_p.A775_G776insYVMA
Mutation 1, KRAS_p.G12C are mutated 1, KRAS_p.G12D mutation 2, KRAS_p.G12S mutation 3, KRAS_p.G12V
Mutation 1, PIK3CA_p.H1047R are mutated 1.Testing result and two generation of Co., Ltd, the medical test institute sequencing of middle source consonance are tied
Fruit is completely the same.
In conclusion the contents of the present invention are not limited in the above embodiments, the knowledgeable people in same area exists
Can propose other embodiments within technological guidance's thought of the invention easily, but this embodiment be included in it is of the invention
Within the scope of.
Sequence table
<110>coordinate Co., Ltd, (Tianjin) medical test institute in source in
<120>primer sets, kit and the application method of lung cancer related gene mutation in mankind's Circulating tumor DNA are detected
<160> 65
<170> SIPOSequenceListing 1.0
<210> 1
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
acgttggatg ttaccttata caccgtgc 28
<210> 2
<211> 27
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
acgttggatg caaccaagct ctcttga 27
<210> 3
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
acgttggatg atcgaggatt tccttgtt 28
<210> 4
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
acgttggatg gaaagttaaa attcccgt 28
<210> 5
<211> 27
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
acgttggatg tctccctccc tccagga 27
<210> 6
<211> 27
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
acgttggatg aggtgaggca gatgccc 27
<210> 7
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
acgttggatg ctgcctcacc tccaccgt 28
<210> 8
<211> 27
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
acgttggatg tttgtgttcc cggacat 27
<210> 9
<211> 29
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
acgttggatg tggtattctt tctcttccg 29
<210> 10
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 10
acgttggatg gcagcatgtc aagatcac 28
<210> 11
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 11
acgttggatg ttataaggcc tgctgaaa 28
<210> 12
<211> 29
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 12
acgttggatg gctgtatcgt caaggcact 29
<210> 13
<211> 29
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 13
acgttggatg ctgaagatgt acctatggt 29
<210> 14
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 14
acgttggatg agtcctgagc ctgttttg 28
<210> 15
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 15
acgttggatg tccagacaac tgttcaaact 30
<210> 16
<211> 31
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 16
acgttggatg ttcatgaaga cctcacagta a 31
<210> 17
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 17
acgttggatg agctcaaagc aatttctaca 30
<210> 18
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 18
acgttggatg tagcacttac ctgtgact 28
<210> 19
<211> 29
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 19
acgttggatg aatccatttt tgttgtcca 29
<210> 20
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 20
acgttggatg aactgagcaa gaggctttgg 30
<210> 21
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 21
acgttggatg gtggggcgcc ccaggcac 28
<210> 22
<211> 31
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 22
acgttggatg cttccttaat gtcacgcacg a 31
<210> 23
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 23
agccctcccg ggcagcgtcg t 21
<210> 24
<211> 17
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 24
gccagcgtgg acaaccc 17
<210> 25
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 25
agctctcttg aggatcttga agg 23
<210> 26
<211> 16
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 26
actcttgcct acgcca 16
<210> 27
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 27
ttagtctgga atgcggactc atgaaa 26
<210> 28
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 28
ttgcgaatac tactcatgat atg 23
<210> 29
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 29
cttctcttaa ttccttgata gcg 23
<210> 30
<211> 16
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 30
ggctttcgga gatgtt 16
<210> 31
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 31
ccgcacccag cagtttggcc 20
<210> 32
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 32
aaacttgtgg tagttggagc t 21
<210> 33
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 33
acttacaggc ttcacccatg act 23
<210> 34
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 34
gtgattttgg tctagctaca g 21
<210> 35
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 35
cttgtggtag ttggagctg 19
<210> 36
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 36
ttgatctttt tgaattcagt tt 22
<210> 37
<211> 16
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 37
tttgggctgg ccaaac 16
<210> 38
<211> 17
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 38
tggtagttgg agctggt 17
<210> 39
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 39
ttaaaattcc cgtcgctatc aa 22
<210> 40
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 40
acaccgtgcc gaacgcaccg gag 23
<210> 41
<211> 17
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 41
agcctacgtg atggcca 17
<210> 42
<211> 16
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 42
ggcactcttg cctacg 16
<210> 43
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 43
tagaaaatct ttctcctgct 20
<210> 44
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 44
gaattcaaaa agatcaaagt gctg 24
<210> 45
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 45
acgtgatggc cagcgtgg 18
<210> 46
<211> 17
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 46
caccgtgcag ctcatca 17
<210> 47
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 47
tggtagttgg agctggtg 18
<210> 48
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 48
tttgttgtcc agccaccatg a 21
<210> 49
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 49
tccactgtgc gacgagctg 19
<210> 50
<211> 17
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 50
acgtgatggc cagcgtg 17
<210> 51
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 51
acgtgatggc cagcgtgg 18
<210> 52
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 52
ttgctgcagc ccaacaact 19
<210> 53
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 53
cgtgtttgcc ttcacgacct gc 22
<210> 54
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 54
tgtattcgcc cgacgtacct gc 22
<210> 55
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 55
caagatcaca gattttgggc 20
<210> 56
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 56
ctgacgaggc gggcagtgtg t 21
<210> 57
<211> 17
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 57
ttggctttcg gagatgt 17
<210> 58
<211> 17
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 58
ctcctgctca gtgattt 17
<210> 59
<211> 17
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 59
agccatggag tacctgg 17
<210> 60
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 60
atgtacagtc ttcccacacg act 23
<210> 61
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 61
ttgctacgtc ccaacaact 19
<210> 62
<211> 17
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 62
tcagcaggtc agagagc 17
<210> 63
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 63
acgttggatg aagttaggtt ttgtcaagaa 30
<210> 64
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 64
acgttggatg cagcaagcag gagtatgacg 30
<210> 65
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 65
gggtccagac tctgcccgtt tggt 24
Claims (9)
1. the primer sets of lung cancer related gene mutation in a kind of detection mankind's Circulating tumor DNA, which is characterized in that primer sets include
PCR amplification primer, Single base extension primer and quality control of procedure primer, PCR amplification primer sequence are SEQ ID No.1-22, single alkali
Base extension primer sequence are as follows: SEQ ID No.23-62, quality control of procedure primer sequence are as follows: SEQ ID No.63-65.
2. detecting the primer sets of lung cancer related gene mutation in mankind's Circulating tumor DNA according to claim 1, feature exists
In the Single base extension primer is divided into 8 groups:
3. the kit of lung cancer related gene mutation in a kind of detection mankind's Circulating tumor DNA characterized by comprising
1) reaction reagent of PCR, including PCR amplification primer described in claim 1, quality control of procedure primer, DNA resistant to high temperature are used for
Polymerase, dNTPs, MgCl2, PCR reaction buffer；
2) reagent for PCR product purifying, including shrimp alkaline phosphotase, shrimp alkaline phosphotase buffer；
3) it is used for the reagent of Single base extension, including Single base extension primer of any of claims 1 or 2, quality control of procedure primer,
Single base extension enzyme resistant to high temperature, the ddNTPs of biotin labeling, extension buffer solution；
4) reagent for extension products capture, the magnetic bead including marked by streptavidin, in conjunction with/washing buffer.
4. detecting the kit of lung cancer related gene mutation in mankind's Circulating tumor DNA according to claim 3, feature exists
In kit further include: negative quality-control product, positive quality control product capture quality-control product, purifying resin, and point sample and Mass Spectrometer Method are used
Target plate.
5. detecting the kit of lung cancer related gene mutation in mankind's Circulating tumor DNA according to claim 3, feature exists
In for assessing the gene mutation site that the mankind suffer from the specificity and sensibility of lung-cancer-risk are as follows: BRAF_p.D594G,
BRAF_p.G469A、BRAF_p.G469V、BRAF_p.V600E、EGFR_p.C797S、EGFR_p.D770_N771insSVD、
EGFR_p.D770_N771insG、EGFR_p.E709A、EGFR_p.E709G、EGFR_p.E709K、EGFR_p.E746_
A750del, EGFR_p.E746_E749del, EGFR_p.E746_S752 > D, EGFR_p.E746_S752 > V, EGFR_
p.E746_T751del、EGFR_p.G719A、EGFR_p.G719C、EGFR_p.G719S、EGFR_p.H773_V774insNPH、
EGFR_p.H773_V774insH, EGFR_p.L747_A750 > P, EGFR_p.L747_E749del, EGFR_p.L747_P753
> S, EGFR_p.L858R, EGFR_p.L861Q, EGFR_p.L861R, EGFR_p.S768I, EGFR_p.T790M, EGFR_
P.V769_D770insASV, ERBB2_p.A775_G776insYVMA, ERBB2_p.G776 > VC, KRAS_p.G12A, KRAS_
p.G12V、KRAS_p.G12D、KRAS_p.G12R、KRAS_p.G12C、KRAS_p.G12S、KRAS_p.G13C、KRAS_
p.G13D、KRAS_p.Q61H、KRAS_p.Q61K、KRAS_p.Q61E、KRAS_p.Q61P、KRAS_p.Q61L、KRAS_
p.Q61R、PIK3CA_p.E542K、PIK3CA_p.E545K、PIK3CA_p.H1047L、PIK3CA_p.H1047R。
6. being detected as described in claim any one of 3-5 in mankind's Circulating tumor DNA the kit of lung cancer related gene mutation
Application method, which comprises the steps of:
1) multi-PRC reaction: in a reaction system, using sample to be tested DNA as template, include institute using being directed to for specificity
The PCR amplification primer for detecting gene mutation site expands the sequence containing different gene locis to be checked by multiplex PCR simultaneously,
Obtain the PCR product of the target area containing amplification；
2) shrimp alkaline phosphatase enzymatic digestion stage 1 is utilized) remaining dNTPs in reaction system；
3) single base extension is carried out using the postdigestive product of Single base extension primer pair step 2), obtains extension products；
4) extension products of saltant type in the magnetic capture single base extension product of marked by streptavidin are utilized；
5) purifying resin, chip point sample, Mass Spectrometer Method are carried out using MassARRAY platform, as a result using TYPER4.0 software point
It analyses and exports as a result, by the way that whether appearance judges mutation type at variation base.
7. detecting the user of the kit of lung cancer related gene mutation in mankind's Circulating tumor DNA according to claim 6
Method, which is characterized in that the PCR reaction condition of the step 1) are as follows: 95 DEG C, 2min；95 DEG C, 30s, 56 DEG C, 30s, 72 DEG C,
1min, 45 circulations；72℃,5min；4 DEG C of holdings.
8. detecting the user of the kit of lung cancer related gene mutation in mankind's Circulating tumor DNA according to claim 6
Method, which is characterized in that the digestion condition of the step 2) are as follows: 37 DEG C, 40min；85℃,5min；4 DEG C of holdings.
9. detecting the user of the kit of lung cancer related gene mutation in mankind's Circulating tumor DNA according to claim 6
Method, which is characterized in that the extension condition of the step 3) are as follows: 95 DEG C, 30s；95 DEG C, 5s, (52 DEG C, 5s, 80 DEG C, 5min)
5 circulations, 40 circulations；72℃,3min；4 DEG C of holdings.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110240999A (en) * | 2018-03-09 | 2019-09-17 | 林云富 | A kind of detection device and method improving Circulating tumor DNA recall rate |
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